Pathogenesis caused by intracellular pathogens, such as Mycobacterium tuberculosis among others, relies on the survival of the microorganism within host cells, such as macrophages and dendritic cells. This mode of infection with or without clinical symptomology hampers bacterial detection, preventing correct diagnosis. Consequently, the physician cannot proceed to the assignment of an appropriate treatment course, delaying the clearance of the pathogen from the body. Serological immunoassays cannot effectively detect intracellular pathogens in biological fluids (e.g. blood, lymphatic fluid) as the microorganism is hidden away within the immune cells. For this reason, isolation of the infected cells and extraction of their DNA, followed by detection of specific bacterial genomic markers via Polymerase Chain Reaction (PCR), is needed to facilitate identification of the pathogen. Although highly specific and sensitive, PCR methods are generally laborious, time-consuming and typically require homogeneous and pure DNA samples [1]. Evidently, complex biological samples, such as biopsies and blood, have to be thoroughly processed in multistep elaborate protocols to obtain pure DNA samples, significantly increasing the final readout time, and affecting the overall DNA yield. An alternative way to detect intracellular pathogens, particularly when they are in low numbers, is by isolating the infected leukocytes from the blood and expanding the number of viable bacteria via culturing methods before PCR analysis. Although very effective, bacterial culturing methods require a significant amount of time, such as in the case of slow-growing intracellular pathogens that have to be cultivated for even weeks, assuming that there is an adequate amount of viable pathogens in the clinical sample. Another drawback of culturing methods is that some pathogens do not grow effectively in culture, either because of the nature of the pathogen itself or the presence of biological interferences present in the clinical sample that prevent growth in culture. Because of these hurdles, new technologies that can detect the presence of a pathogen in human clinical samples are urgently needed.
It has been predicted that nanotechnology will have a major impact in medicine, agriculture and biotechnology among other fields [2], [3], [4], [5], [6], [7], [8], [9], [10]. Innovative technologies that take advantage of the unique size-dependent electronic, magnetic and luminescence properties that some nanomaterials exhibit when they specifically interact with a biological marker have been developed for faster and more accurate sensing of various pathogens [2], [3], [4], [5]. However, the translation of these technologies to the clinical diagnosis of an infectious disease and how it relates to the presence of the pathogen in clinical samples has been limited. The slow clinical translation of some of these nanotechnologies has been partly due to their poor performance in crude or minimally processed clinical samples. Among the most promising nanomaterials are magnetic relaxation nanosensors (MRS). These magnetic nanosensors are composed of a polymer-coated iron oxide nanoparticle onto which affinity ligands are conjugated to facilitate binding and magnetic detection of a particular target [11], [12]. Upon specific binding of a target to ligands on the magnetic nanoparticle, changes in the sample's magnetic resonance signal (specifically the water proton relaxation time; T2) occur that correlate with the target concentration in solution. By measuring the changes in T2 relaxation times upon target interaction and correlating the intensity of the change with the target concentration, one can develop a sensitive detection method, therefore the acronym of magnetic relaxation nanosensors (MRS).
In contrast to other methods that utilize magnetic nanoparticles as labels and directly measure an intrinsic physical property, such as a magnetic field [13], [14], or a chemical property [6], the MRS method relies on the effect that the nanoparticle's induced magnetic field exerts on the hundreds of thousands of water molecules surrounding the nanoparticle. This effect results in an amplification of the signal, allowing for sensitive detection even in turbid and minimally processed samples, without the need of further amplification, which is critical for assays such as PCR and ELISA. Hence, MRS-based assays can be more affordable than traditional assays, since they use a single reagent that can be easily produced in large quantities, and a single-step that avoids time- and labor-consuming procedures. Additionally, MRS can detect their targets at various point-of-care settings, since the nanosensors are stable at ambient conditions and utilize deployable instrumentation. Recently reported is a unique MRS nanoparticle-target interaction that resulted in rapid T2 increases upon target binding [12]. This unique nanoparticle-target interaction is different from other MRS-based methods, because clustering of the nanoparticles is not required to achieve detection. In contrast, binding of MRS to the target caused fast, target-concentration-dependent increases in T2. Kinetic studies also revealed that the binding of MRS was faster and more sensitive than the MRS clustering, while achieving lower detection thresholds. In addition, nanoparticle valency—the amount of a targeting ligand on the surface of the nanoparticle—plays a role in the magnetic relaxation response. Nanoparticle valency controls the trend of the change in the spin-spin relaxation time (ΔT2). Specifically, it has been found that low valency results in decreases in the ΔT2 as the target concentration increases, while at high valency conditions the ΔT2 increases as the target concentration increases.